In this application, we propose to develop a novel technique, namely, RNA-guided RNA 2'-O-methylation, for regulating telomerase activity in vivo. We also propose to understand how 2'-O-methylation, as a naturally occurring process, is adopted by cells to trigger the cessation of growth under certain conditions. Given the close relationship between telomerase activity and aging, we believe that our proposed work (under three specific aims, see below), once accomplished, will significantly advance our understanding of telomerase regulation, aging, and human diseases. Specific Aim 1--Targeting of nucleotides in the conserved pseudoknot region of TLC1 Using box C/D RNA-guided 2'-O-methylation, we have artificially targeted TLC1 at six sites within the conserved pseudoknot region, and identified at least three sites where modification led to altered telomerase activity (two led to enhancement and one to reduction). Building on these results, we plan to expand our modification targets to cover each and every site in this important pseudoknot region. Any sites at which 2'-O- methylation affects telomerase activity will be identified and selected (individully or in combination) for further telomerase activity analyses, telomerase RNP assembly and recruitment assays. Our studies will allow us to understand the basis of effects, thus offering a variety of choices to influence telomerase activity and aging. Specific Aim 2--Global screening of important 2'-OH groups in TLC1 via unbiased 2'-O-methylation Given that TLC1 is a long RNA molecule and has at least several important domains, we believe that some nucleotides (and their 2'-OH groups) outside of the pseudoknot region are important for function as well. To obtain a complete spectrum of the effects of 2'-O-methylation, we propose an unbiased full screen throughout the TLC1 sequence. Specifically, we will use TLC1 cDNA to construct an artificial box C/D guide RNA library, directing 2'-O-methylation to each and every nucleotide of TLC1. A growth phenotype screen assay will be carried out to identify important sites where 2'-O-methylation negatively impacts telomerase activity. Specific Aim 3--Identification of enzyme responsible for induced TLC1 2'-O-methylation Our preliminary results also showed that upon entry into the stationary phase, TLC1 was 2'-O-methylated (~50%) at several sites, including a site where modification reduces telomerase activity. This remarkable result prompted us to explore further the mechanism of this inducible 2'-O-methylation. Specifically, we propose to identify the enzyme responsible for this induced modification. To achieve this goal, we plan to conduct a series of experiments, including screening using yeast ORF libraries (if the activity is orchestrated by protein only) or a yeast box C/D guide RNA library (if a box C/D RNP is involved). Identification of the enzyme responsible for induced 2'-O-methylation will eventually lead to the elucidation of the mechanism of this modification, and will thus significantly advance our understanding of the regulation of telomerase activity, aging and diseases.